1. Field of the Invention
This invention relates generally to the field of biotechnology, and, more specifically, to a process for detecting specific molecules in an in vitro system by the detection of a signal produced by a living cell or selectively grown cells.
2. Prior Art
A number of prior methods have been developed and used extensively for the detection of specific molecules, such as DNA, RNA, protein, peptides, carbohydrates, fats, minerals, various organic and inorganic molecules, including complexes of the same. For example, in the area of basic biological research, the identification of specific chains of DNA and RNA is important in the determination of the genetic makeup of cells, and the isolation and purification of nucleic acids for recombinant DNA technology.
In the field of medical diagnostic testing and research the diagnosis of certain genetic diseases requires the identification of specific DNA segments. Some of these assays use DNA restriction-fragment-length-polymorphisms (RFLP) as tools for diagnosis of genetically linked diseases in which DNA of affected individuals is analyzed using Southern blotting to reveal a particular restriction fragment length cf DNA in close genetic linkage to a gene defect.
As other examples, the detection of cancer, the evaluation of the progress of cancer treatment drugs on such cancers, and the characterization of tumors is dependent on the ability to detect and measure the level of various blood factors, hormones, drugs, proteins and other biological molecules in the blood and other body fluids and tissues. On therapeutic drug monitoring, See Human Pathol, 15:404-411 (1984); on assays for drugs, hormones, proteins and antigens, including viral and bacterial antigens, see Human Pathol. 15, 112-116 (1984); on diagnosis of infectious disease, see Ann. N.Y. Acad Sci 428:223-229 (1984).
In the prior art, there are essentially three types of systems for the detection of specific molecules. The first method, and the one that has been used most extensively in recent years, involves the use of radioactive molecules (radio immune assays or RIAs; Southern, Northern and Western blots etc.) A second method, which in general is less sensitive, is the use of enzymatic reactions. The third method, which is generally least effective because of its relative insensitivity and requirement for very detailed technical work, is the use of physical detection methods, such as infra-red spectrum, NMR spectrum, UV absoption, fluorescence and the like.
With respect to radioactive probes, it is well-known that the more sensitive the probe is, the more radioactive it must be. Thus, highly radioactive molecules, such as those containing iodine-125 or phosphorous-32 isotopes decay relatively rapidly so that the radioactive decay can destroy the probe designated to identify the target molecule. Furthermore, in view of the rapid radioactive decay, these radioactive probes have a short shelf life. Moreover, they are hazardous to work with and difficult to dispose of, as a result of the innate problems involved with a dangerous radioactive material.
In enzymatic systems, an enzyme is chemically coupled to a molecule that is capable of specifically binding with a target molecule which is to be detected. The enzyme thereafter is induced to produce a signal by acting on a substrate to convert it into a detectable colored molecule, or result in the production of light of a particular wave length, and the signal is detected by known means.
Unfortunately in both the radioactive and enzymatic signal systems, one probe is capable of producing only a limited signal. That is, the molecule (i.e. the probe) that is capable of binding to the target molecule can only attach to or react with a single or a very small number of signal producers. If the signal is a radioactive probe, such as phosphorous-32, only a limited number of phosphorous-32 molecules can be bound to the probe. Each individual phosphorous-32 molecule can only emit a single signal upon disintegration; that is, each phosphorous-32 molecules can only decay once in a manner that will be detected by a radioactive counter, or by autoradiography. Similarly, with respect to an enzymatic signal system, there is a limited amount of substrate with which a single enzyme molecule coupled to a probe may catalyze a reaction within a specified time period to thereby produce a signal indicating detection of the target molecule. Another problem with this system is that the product of the enzymatic reaction is often designed to be relatively insoluble in order to limit diffusion of the signal, thus limiting substrate access to the enzyme.
3. Definitions
The following terms will be used herein and are defined as follows to facilitate understanding of this patent. The definitions provided are not intended to limit in any way the scope of this patent or restrict the definitions to be narrower than is understood in the field.
Amplify--to increase the level of a signal denoting the presence of a target molecule with the intention of making the signal detectable. Amplification is particularly useful when the target molecule is only available in very low concentrations.
Lysogenic bacteria--Bacteria which contain a phage genome incorported within the bacterial genome. The incorporated phage genome produces a repressor molecule which inactivates its own gene systems as well as inactivating similar phages (upon which the repressor has a proper binding site) which enter that bacteria.
Phage (bacteriophage)--a virus comprising a protein coat and nucleic acids. The phage is capable of attaching to a bacterial cell and injecting its DNA (or RNA) into the cell. Thereafter, the phage may use the cell's synthesis abilities to reproduce copies of the phage's genome and protein coat, which combine and subsequently are extruded or burst out of a cell, and which, in turn, are then able to infect other cells. Alternatively the phage may innocuously replicate its DNA within its host bacterias' DNA and latently infect the bacterial cell's progeny (in the case of lysogenic bacteria).
Phagemid--a combination of a phage and a plasmid. A phagemid can infect a bacteria like a phage, but in a lysogenic bacteria can only replicate by using the plasmid origin of replication, in which case the phage behaves like a plasmid with essentially all of the phage genes turned off, so that only the plasmid genes are expressed.
Plasmid--a circular piece of DNA which can replicate and maintain an independent existence in bacterial cytoplasm, separate from the bacterial chromosome.
Probe--a molecule that can bind, by means of ionic, steric or other known forms of interaction, to another specific or "target" molecule. Antigen-antibody, enzyme-substrate, DNA and RNA complements are examples of possible probes binding to specific molecules.
Replication--the duplication of a DNA molecule to make a copy of the same. Replication occurs when a cell divides so that both daughter cells have the same DNA content.
Signal--any message which is intended to be detected, thereby indicating the presence of a target molecule.
Southern Blotting--method of detecting the existence of specific DNA fragments. The target DNA fragments are bound to a substratum and the probe DNA or RNA fragments are radioactively labelled and then hybridized to the bound fragments. The successfully hybridized fragments (indicating a match between target and probe) are then detected, generally by autoradiography. See Meinkoth and Wahl, "Hybridization of Nucleic Acids Immobilized on Solid Supports, Anal. BioChem. 138, 267-284 (1984); Wahl et al, "Efficient transfer of large DNA fragments from agarose gels to diazbenzylomethyl-paper and rapid hybridization by using dextran sulfate," Proc. Nat. Acad. Sci. U.S.A., 76:3683-3687 (Aug. 1979); and Southern, "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis," J. Mol. Bio. 98: 503-517 (1975).
Substratum--any immobilized material or surface which immobilizes the target.
Tag--any chemical linkage between two molecules.
Target or Target Molecule--any molecule which is intended to be detected.
Transfection--the act of a virus (phage) injecting its DNA (or RNA) into a host cell.